Gonadotropin-releasing hormone (GnRH) drives the hypothalamic-pituitary-gonadal axis. The GnRH neurons have an extraordinary origin. In the mouse, they arise in the presumptive vomeronasal organ within the olfactory placode at e11.5. As they differentiate, they migrate past the cribriform plate, through the olfactory bulb and into the hypothalamus, finally extending their axons to the median eminence. This discrete population of ~800 neurons, scattered through the hypothalamus, controls puberty, menstrual cyclicity, fertility, and meno- pause. Disrupted migration or disregulated secretion results in failures in puberty, fertility, and reproductive function. The overall goal of this renewal application is to elucidate the molecular, epigenetic, and developmental mechanisms that regulate hypothalamic GnRH neuron migration and maturation. We will utilize two major model systems: our immortalized GnRH-secreting hypothalamic cells (GT1) and genetically modified mice. Under the support of this long-standing grant, we have identified evolutionarily conserved promoter and enhancer regions that target GnRH gene expression exclusively to cultured GT1 cells in vitro and to GnRH neurons in vivo. We have shown that these GnRH regulatory regions are controlled by transcriptional regulators: Oct1, NF1, Gata4, Otx2, Pbx, Prep, Meis, Dlx, Msx, necdin, C/EBP, Six6/3, and the TLE (Grg) co- repressors, and that mouse models lacking any one of a number of these fail to produce the correct population of GnRH neurons and/or disrupt migration in vivo. For this renewal application, we propose three novel aims: Aim 1 will address the differentiation and maturation of the GnRH neuron during migration determining the fate of neurons that lack expression of factors in the Dlx/Msx/Necdin pathway, and elucidate the mechanism of Msx repression of GnRH gene expression. Aim 2 will focus on specific homeodomain factors crucial for the development of the hypothalamus as a whole and determine their role in GnRH neuron migration and maturation in vivo and in vitro. Aim 3 will address the fundamental mechanisms of the neuron-specific enhancers of the GnRH gene. We have discovered that the major enhancer critical for neuron-specific GnRH expression is bound by activated Polymerase II and is transcribed into RNA. This long, noncoding RNA will be characterized in vitro and in vivo and its mechanisms of action will be determined. The homologous enhancers in human will be analyzed and screened for mutations in hypogonadal patient DNA. Our overarching hypothesis is that developmental migration and maturation of the GnRH neuron require the acquisition of a transcriptional regulatory program that relies on developmental coordination of homeodomain transcription factors and long non-coding enhancer RNA. We believe these studies will provide valuable insight into novel regulatory mechanisms and enable progression beyond the currently accepted paradigms. This multifaceted approach should yield a comprehensive understanding of the program of GnRH neuronal migration and cell fate in vivo and in vitro and provide insight into the genetics of hypogonadotropic hypogonadism.